Fig. 4
miR-1199-5p directly controls Zeb1 expression. a Species-conserved miR-1199-5p-binding site in the 3′ UTR of Zeb1. The scheme represents: top: sequence alignments of predicted binding sites of miR-1199-5p (red; mouse: position 239–246) in Zeb1 3′ UTRs of different species. Middle: alignment of the 8mer seed match in mouse miR-1199-5p. Bottom: mutated miR-1199-5p seed sequence in the 3′ UTR of Zeb1 mRNA utilized for the control reporter construct (Zeb1 3′ UTR mut) in d. Exchanged nucleotides are underlined. b, c Forced expression of miR-1199-5p reduces Zeb1 nuclear localization and protein levels during an EMT. NMuMG/E9 cells were transiently transfected with miRNA mimics as indicated and cultured in the absence (0 day) or presence of TGFβ (4 days). Immunofluorescence (b) and immunoblotting (c) analyses illustrate the differences in Zeb1 protein levels. Scale bar: 100 μm. d Post-transcriptional regulation of Zeb1 by miR-1199-5p. NMuMG/E9 cells were transfected with the miRNA mimics indicated, with a Renilla luciferase reporter construct and with either a Zeb1 3′ UTR wild-type (wt) or a Zeb1 3′ UTR mutant (mut) Firefly luciferase reporter construct. Relative luminescence (Firefly/Renilla) was calculated and normalized to miR-Ctr-transfected cells (mean fold changes±s.e.m.; n = 3; significance determined by an unpaired, two-sided t test; *P < 0.05)
